Cells have evolved complex protein quality control systems comprised of multiple chaperone and clearance pathways to handle both misfolded non-amyloidogenic and amyloidogenic proteins. To better understand the mechanisms used to eliminate amyloids, we have been using yeast as a model organism due to the high conservation of cellular pathways from yeast to man plus it has the advantages of a short generation time and easy genetic manipulation. The amyloids we have studied include naturally occurring prions and mammalian huntingtin fragments with expanded polyglutamine repeat region. Prion proteins, which have a properly folded conformation and a misfolded amyloid conformation, exist as cytoplasmic seeds. These seeds, which are severed by the molecular chaperone Hsp104, are transmitted from mother to daughter cells as the yeast divides. To cure yeast of the misfolded amyloid form of the prion, all the prion seeds must be eliminated, which can occur by several different methods. Prions are cured when severing of the prion seeds is inhibited; in the absence of new prion seed production, the prion seeds are diluted out by cell division. Another mechanism of curing yeast prions is aggregation of the prion seeds followed by asymmetric segregation of the seeds between mother and daughter cells, thus reducing the number of seeds transmitted to the daughter cells. Finally, yeast prions can be cured by dissolution of the seeds, which can occur either by dissociating monomers from the ends of the seeds or by inhibiting growth of the seeds. To differentiate the mechanism by which different proteins cure prions, we have combined real-time imaging of fluorescent-labeled prion proteins with measuring the time course of curing using plating assays. Our research on the curing of PSI+ by Hsp104 overexpression has supported a model in which there is dissolution of the prion seeds that is dependent on the trimming activity of hsp104. Trimming activity of Hsp104, unlike severing activity, makes the seeds smaller, but does not increase the number of seeds. We examined the relationship between the trimming and the curing of PSI+ by Hsp104 overexpression over a wide range of conditions including using different Hsp104 constructs, different levels of Hsp104 overexpression, and different PSI+ variants. Weak variants, which have fewer seeds and more soluble Sup35 than strong variants, not only are cured at a faster rate by Hsp104 overexpression, overexpressed Hsp104 trims the weak PSI+ variants at a faster rate than the strong variants, In addition, we find that the Hsp70 protein, Ssa1, not only inhibits curing by Hsp104 overexpression, it inhibits trimming of the prion seeds as well. Conversely, we find that a Ssa1 with a point mutation at L485W increases the trimming activity of Hsp104, as well as increases the rate of curing of PSI+ by Hsp104 overexpression. Moreover, overexpression of this mutant Ssa1 in a weak PSI+ variant trims the prion seeds at endogenous levels of Hsp104 and concomitantly cures the PSI+ prion. Our research on URE3 has focused on the molecular chaperone Hsp42. Overexpression of Btn2, Hsp42, Cur1, or Ydj1 in URE3 yeast produces aggregation of the Ure2 seeds, which leads to curing by asymmetric segregation. By expressing these proteins in an HSP42 deletion strain, we find that the Ure2 seeds no longer forms large aggregates. Whereas overexpression of Btn2 does not cure URE3 in an HSP42 deletion strain, overexpression of either Cur1 or Ydj1 still cures URE3, but at a significantly slower rate. These proteins now cure URE3 primarily by inhibiting the severing activity of Hsp104. To better understand the role of Hsp42 in aggregation of Ure2 foci, we have expressed endogenous levels of fluorescently labeled Hsp42 in URE3 cells. When Btn2, Cur1, Ydj1, or Hsp42 is overexpressed, the diffuse cytosolic Hsp42 assembles into large clumps that colocalize with aggregated Ure2 foci. Aggregation of the Ure2 seeds by Hsp42 only occurred with the full-length Hsp42; truncating either the N-terminal prion domain or the intrinsically disorder domain of Hsp42 eliminated aggregation of the Ure2 seeds. We conclude that Hsp42 acts as a scaffold that tethers the Ure2 seeds, which in turn leads to the curing of URE3 by asymmetric segregation. In a related project, we are examining the aggregation of huntingtin (Htt) exon 1 fragments in yeast, which has been used as a system for studying Huntingtons disease. This neurodegenerative disorder is caused primarily by the accumulation of Htt exon 1 fragments in the brain. Htt exon1 fragments consist of three regions, an N-terminal region of 17 amino acids, a polyQ region of variable length, and a C-terminus that is rich in proline. In yeast as in mammalian cells, aggregation of Htt fragments is dependent on the length of the polyglutamine repeat region. However, unlike mammalian cells, Htt fragment aggregation has been reported to be dependent on the presence of a yeast prion. This, in turn, makes aggregation dependent on Hsp104, which by severing the prion seeds enables them to propagate. It is not clear whether Hsp104 has another role in Htt aggregation other than maintaining the yeast prion. Therefore, we have expressed Htt fragments with expanded polyglutamine repeat regions without and with the polyproline region (HttpolyQ and HttpolyQP fragments) in yeast with prion. Surprisingly, the HttpolyQ aggregates, which form numerous aggregates per cell, persist in yeast even after the prion is cured provided that Hsp104 is active. However, the HttpolyQP aggregates, which forms one large aggregate or aggresome per cell, do not persist in cured cells. These aggregates are severed by Hsp104, which enables them to be maintained in the absence of prion. In fact, we find that HttQ103 aggregate formation, but not HttQP103, occurs in the absence of prion provided the cells express Hsp104. Although prion is not necessary for HttQ103 aggregation, it greatly accelerates the rate of aggregation formation. These results have redefined the role of prion and Hsp104 in HttpolyQ aggregation in yeast.